CN108948172B - 一种仿生医用成丝纤维蛋白的制备及其应用 - Google Patents
一种仿生医用成丝纤维蛋白的制备及其应用 Download PDFInfo
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Abstract
本发明提供一种成丝纤维蛋白的,其序列为SEQ ID NO:1。本发明在一个方面提供一种用于制备成丝纤维的蛋白片段的制备方法,所述的蛋白片段是序列为SEQ ID NO:1的扇贝足丝蛋白的160‑324位氨基;其序列为SEQ ID NO:2。本发明制备成丝纤维的蛋白在材料来源上采用基因工程法制备,成本低廉,绿色环保可大规模生产。本发明的蛋白基材料来源于海洋扇贝足丝,并在类似海水环境中聚合成形,对湿环境具有良好的适应性和稳定性。在医用材料领域具有较好的开发前景。在材料生物安全性上,海洋生物来源材料避免了陆地生物疾病传播的风险,经检验材料无细胞毒性,具有生物相容性好。
Description
技术领域
本发明属于医学材料制备技术领域,具体涉及一种成丝纤维及其应用。
背景技术
生物医用材料(Biomedical Materials)也称为生物材料(Biomaterials),是一种能对机体的细胞、组织和器官进行诊断、治疗、替代、修复、诱导再生或增进其功能的特殊的功能材料。生物机体内含有大量的以水为基础的细胞外液,机体细胞组织更是始终处于由组织液,血浆和淋巴液组成的“湿”环境中。因此用于体内修复,封堵,支持材料首先必须具有良好的水下稳定性和溶胀性,更高层次上还要求优良的生物相容性和可控的生物降解吸收性。目前主要用于体内环境的热门研究材料主要包括一些细胞外基质(extracellularmatrix,ECM)蛋白如胶原和弹性蛋白,多糖类如壳聚糖,海藻酸盐,透明质酸等材料。这些材料虽然有自身独特的应用优势但也伴随着许多的缺陷。例如天然的胶原蛋白水凝胶机械性能较弱,降解速度较快和容易引起免疫反应等缺点;天然透明质酸对强酸、强碱、热、自由基及透明质酸酶敏感稳定性差,容易发生降解;壳聚糖由于晶体结构紧密,无法溶于中性溶液和大多数有机溶剂,因而其应用受到极大限制。目前新型体内外“湿”环境应用材料的开发仍是目前研究热门领域。
发明内容
本发明提供一种成丝纤维蛋白的制备及其应用,从而弥补现有技术的不足。
本发明首先提供一种扇贝足丝蛋白,包含有:
1)序列为SEQ ID NO:1的蛋白;
2)从海洋扇贝足丝中获得的,与1)中的序列具有80%以上同源性,且具有1)中蛋白功能的蛋白;
上述的2)中同源性,是指具有90%或以上的同源性,最优选为具有99%的同源性;
本发明在一个方面提供一种用于制备成丝纤维的蛋白片段的制备方法,所述的蛋白片段是序列为SEQ ID NO:1的扇贝足丝蛋白的160-324位氨基;其序列为SEQ ID NO:2;
本发明的序列为SEQ ID NO:1和SEQ ID NO:2的蛋白片段在制备临床医用材料中的应用;
所述的临床医用材料,包括手术缝合线、导管、纤维类材料或组织工程支架材料;
本发明还提供一种用于制备成丝纤维的蛋白液,是将序列为SEQ ID NO:2;的蛋白溶解于六氟异丙醇(HFIP)中制成的;
所述的蛋白液中蛋白的浓度,优选为150mg/mL。
本发明制备成丝纤维的蛋白在材料来源上采用基因工程法制备,成本低廉,绿色环保可大规模生产。目前正在应用材料,包括如胶原,弹性蛋白和壳聚糖等多来源于从生物体及其产物中提取。这种方法制取原料昂贵,工艺繁琐复杂且大多都有污染。而且材料成形上为仿生“湿”环境成形,材料成形后在体内外“湿”环境形态结构稳定,溶胀性较低。胶原,壳聚糖等材料成形一般在有机试剂或无水环境中,在体内外“湿”环境应用时由于溶胀性原因形态结构会发生变化。由于Sbp5-2蛋白基材料来源于海洋扇贝足丝,并在类似海水环境中聚合成形,对湿环境具有良好的适应性和稳定性。在医用材料领域具有较好的开发前景。在材料生物安全性上,海洋生物来源材料避免了陆地生物疾病传播的风险,经检验材料无细胞毒性,具有生物相容性好。
附图说明
图1:Sbp5-2功能模块划分及克隆模块示意图;
图2:RM6CT基因的扩增产物凝胶电泳图;
图3:纯化后蛋白SDS-PAGE电泳图片:
图4:RM6CT蛋白成纤维图片A为自然环境中扇贝足丝形态,B为RM6CT蛋白拉丝成形过程中的形态,C为RM6CT蛋白拉丝稳定后的人工丝形态。
图5:RM6CT蛋白纤维溶胀率(左)和吸水率(右)检测结果图;
图6:RM6CT蛋白纤维湿环境下光学纤维镜下形态观察。刚成丝15min(左),成丝后48h(右);
图7:细胞毒性评估图。
具体实施方式
申请人发现,扇贝足丝长期浸泡的海水环境中包含较多的材料腐蚀因素如海水含盐量,温度,溶氧量,pH值,流速与波浪,海洋微生物等,在如此恶劣的环境,扇贝足丝依旧可以保持稳定的形态结构和力学性质。申请人认为扇贝足丝蛋白具有在体内外“湿”环境应用材料领域的开发潜力和价值。
根据这些性质的初步研究分析Sbp5-2蛋白丝在临床医用材料等多个领域,包括手术缝合线、导管及纤维类材料的制备以及作为组织工程支架材料等。
实施例1:Sbp5-2蛋白的筛选
1、Sbp5-2全基因的克隆
1.1结合质谱鉴定结果序列信息,设计RACE引物
运用Primer Premier 5软件设计RACE(Rapid Amplification of cDNA Ends)Sbp-5-2的引物
3’RACE引物:TCCCAACAATGGAGTATGTGAAGATGCTG、
5’RACE引物:GCCTAGTGTAACAGTCATTCTCCAGCCAAG;
1.2 RNA抽提
取刚切断足丝时足适应期(0-2h)、足丝分泌增长期(2-16h)以及足丝分泌平衡期(16-24h)的三只栉孔扇贝进行解剖后取其足,依照Hu等(2006)的方法抽提RNA并等量混匀。利用SMARTer TM RACE cDNA Amplification Kit(Clontech,CA,USA),按照说明,构建5’RACE和3’RACE文库。
1.3分别进行目的基因的3’和5’末端的克隆
在PCR管中加入:
混匀后置于PCR仪中按以下程序进行扩增:
1.4切胶回收克隆测序
用1%的琼脂糖凝胶进行检测后,1×TBE配制2%的低熔点琼脂糖胶,电泳缓冲液为1×TBE,将样品全部点到胶孔后,在100V电压下电泳45min,切胶产物用QIAquick GelExtraction Kit进行标准纯化,产物溶于三蒸水中,与
pMD18-T载体进行连接,转化到大肠杆菌DH5α,进行菌落PCR,挑取目的片段插入成功的阳性克隆进行Sanger测序,利用Star对3’和5’末端序列进行拼接获得Sbp5-2目的基因cDNA全长序列,其氨基酸序列为SEQ ID NO:1。
MYAVYLFVAVFILPIILAANDECTSKGQCTALDGVTCVSLGQQRLEKCNTYTCRRSNNVLKYKIVKNLLKCKRPDGTSMDIGVGEKDETKCTTEICRRARNSDGTFTLTYREKPYGCPLTDGSCLLFGKRNKIRNENKCLDTTCTRRKNKKGQYISRLKNKYYGCPNNGVCEDAEATRNNSCTSYMCVLSKRRTLMKWEILKTGCKTDEGCKYDTDEWTDPDASSCVTRRCDVTFNPTDKTYNSVNRVARHGCRASNGTCYYNSETWLENDCYTRRCDVTTTDKGESMAAIKIESGICKDADGSCKGYGEAMQYRSGAATLNCVCDEAISTQGYPQGRPVCKSP
实施例2:成丝纤维的制备
一、RM6CT的克隆表达
根据Sbp5-2氨基酸序列分析发现除去信号肽序列的部分具有多个以成对半胱氨酸形成的功能模块,以及不同于功能模块的C末端(C Terminal)序列。功能模块中包含众多的亲水性氨基酸经筛选选择C末端(C Terminal)序列后连接的六个功能模块的亚克隆RM6CT(氨基酸序列号:160-324)进行纯化表达。(图1),所述的亚克隆RM6CT的序列为SEQ IDNO:2;
NKYYGCPNNGVCEDAEATRNNSCTSYMCVLSKRRTLMKWEILKTGCKTDEGCKYDTDEWTDPDASSCVTRRCDVTFNPTDKTYNSVNRVARHGCRASNGTCYYNSETWLENDCYTRRCDVTTTDKGESMAAIKIESGICKDADGSCKGYGEAMQYRSGAATLNCV;
1.1 RM6CT引物设计
5’引物:GGGGTACCAACAGTTGCACCTCATATATGTGTA
3’引物:CCGCTCGAGTTACGGACTTTTACACACTGGT
1.2进行RM6CT的扩增PCR
在PCR管中加入:
混匀后置于PCR仪中按以下程序进行扩增:
核酸琼脂糖凝胶电泳如图2所示。
1.3 RM6CT工程菌表达
将PCR产物使用核酸纯化试剂盒进行纯化,然后分别使用KpnI和XhoI内切酶将PCR产物和PET32-Kan载体质粒进行双酶切。按照PCR产物和载体质量比10:1的比例进行连接。转化进入工程大肠杆菌BL21(DE3)。
1.4 RM6CT的表达纯化
(1)将转化后携带RM6CT基因的工程菌培养在LB培养基中,待生长至OD600为0.6-0.8时,加入0.25mM的IPTG。诱导培养在37℃,180rpm震荡培养10h。离心机离心菌液收取菌体。
(2)取2g离心收集的菌体加入20mL PBS缓冲液重悬。冰水浴下超声破碎细胞:使用6号变幅杆,作用5s,间隔10s,功率60%,超声时间为35min。12000g,4℃离心15min,弃去上清留取沉淀。
(3)分别使用以下溶液重悬沉淀,冰水浴下超声洗涤纯化:使用6号变幅杆,作用3s,间隔6s,功率60%,超声时间为15min。12000g,4℃离心15min,弃去上清留取沉淀。
20mL PBS缓冲液洗涤沉淀(重复两次)
20mL 1M尿素/1%甘油/1%TritonX-114洗涤(重复两次)
(4)使用20mL 8M尿素/20mM Tris-HCl(pH8.5)/10mM DTT变性缓冲液将蛋白沉淀溶解。12000g,25℃,离心15min,收集上清。
(5)透析复性:将溶解的蛋白溶液稀释至1mg/mL,装入截留分子量为8000-14000Da的透析袋中,依次透析在20mM,10mM,5mM,1mM和0mM的Tris-HCl(pH=8.5)/1mM DTT/透析液中。重复透析几次直至完全除去溶液中的尿素和Tris-HCl。透析后溶液分装于10mL离心管,液氮中冷冻后-80℃保存。
纯化后蛋白SDS-PAGE电泳图片如图3所示。
二、RM6CT仿生成丝工艺
2.1成丝液制备
将液氮冰冻保存的RM6CT蛋白溶液,在冻干机中进行冻干处理获得RM6CT冻干粉。将RM6CT冻干粉按照150mg/mL的浓度溶解于六氟异丙醇(HFIP)中,室温摇荡促溶,直至溶解均匀。
3.2仿生成丝
取10μL成丝液,打入类似海水环境的20mM Tris-HCl(pH8.5)/10mM CaCl2溶液中,静置1-2s,挑起聚集蛋白拉伸成丝。在20mM Tris-HCl(pH8.5)/10mM CaCl2中稳定10min,可获得在湿环境下较为稳定,并具有一定弹性的蛋白人工丝(图4)。如图4所示,该纤维蛋白丝可以制备成任意的长度,具有的机械强度(如图4-C),提示其未来在医用材料领域具有广阔的应用前景。
实施例3:成丝纤维的性质
1、成丝纤维的基本性质表征:
1)材料湿稳定性和吸水率检测
将宏观成形湿态的纤维丝(约0.1g)浸泡于PBS溶液中,分别于0,12,24,36,48,60,72,84,96,108h时取出用滤纸吸干表面的水分,称质量为Mwet。放-80℃冰冻后进行冻干处理,冻干后称其质量为Mdry,做三组平行。
溶胀率=(Mwet-Mdry)/Mdry×100%。
吸水率=(Mwet-Mdry)/Mwet×100%。
图5为RM6CT蛋白纤维溶胀率(左图)和吸水率(右图)检测结果图;检测结果说明在湿环境下保持较稳定的溶胀性能,108小时内材料溶胀率维持在400%左右。且保持80%左右的吸水率;
2)光学显微镜观察湿环境下Sbp5-2蛋白基纤维材料形态
通过光学显微镜观察经过PBS溶液浸泡15min和48h后的蛋白纤维形态,其形态都为比较致密的充盈结构。48h内Sbp5-2蛋白纤维形态几乎无变化,证明Sbp5-2材料具有良好的水下稳定性和较高的吸水率。揭示其在生物医用材料领域的巨大开发潜力(图6)。
3、细胞毒性评估
将纯化的RM6CT蛋白分别按照0.5,1.0,2.0,5.0,10,20和40ng/mL的浓度与3000个小鼠成纤维L929细胞孵育24h,48h和72h。MTT法检验细胞毒性。
MTT检测结果表明各浓度蛋白与小鼠成纤维L929细胞孵育24h,48h和72h后MTT结果与空白对照相比均无显著差异。表明RM6CT无细胞毒性作用(图7)。
序列表
<110> 中国海洋大学
<120> 一种仿生医用成丝纤维蛋白的制备及其应用
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Claims (5)
1.一种用于制备成丝纤维的蛋白片段,其特征在于,所述的蛋白片段是序列为SEQ IDNO:1的扇贝足丝蛋白的160-324位氨基酸;其序列为SEQ ID NO:2。
2.权利要求1所述的蛋白片段在制备临床医用材料中的应用,所述的临床医用材料包括手术缝合线、导管、成丝纤维或组织工程支架材料。
3.一种用于制备成丝纤维的蛋白液,其特征在于,所述的蛋白液是将序列为SEQ IDNO:2的蛋白溶解于溶剂中制成的,所述的溶剂为六氟异丙醇。
4.如权利要求3所述的蛋白液,其特征在于,所述的蛋白液中蛋白的浓度为150 mg/mL。
5.权利要求3或权利要求4所述的蛋白液在制备成丝纤维中的应用。
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